Until recently molecular biology has not been applied to investigations involving P. carinii. This has been due to the difficulty in working with P. carinii organisms and obtaining them in large quantities in a purified form. Using the animal model of P. carinii pneumonia, we are able to obtain large quantities of the organism, but are unable to purify them from host lung cells. Such partially purified P. carinii have been utilized in studies to investigate the chromosomes of P. carinii by pulse field gel electrophoresis, a technique that allows separation of large molecular weight chromosomes. Using this technique we have identified a minimum of 15 chromosomal sized DNA bands from P. carinii. Looking at a variety of rat P. carinii isolates we have been able to identify differences in the sizes of the chromosomes of P. carinii among different isolates. In addition we have identified, using Southern blots and DNA hybridization, the chromosome on which the genes for P. carinii dihydrofolate reductase, thymidilate synthase, and ribosomal RNA reside. These studies will be useful in developing a genetic map of P. carinii and in confirming that clones which have been identified are in fact specific for P. carinii.